Microvascular free tissue transfer for gene delivery: in vivo evaluation of different routes of plasmid and adenoviral delivery

Abstract

Transfer of healthy autologous tissue as a microvascular free flap facilitates reconstruction during ablative cancer surgery. In addition to filling surgical defects, free flaps might concentrate viral vectors at the tumour bed and mediate local therapeutic effects. We evaluated the magnitude, topography and duration of luciferase gene expression after plasmid and adenoviral delivery in rat superficial inferior epigastric (SIE) flaps. For plasmid delivery, luciferase expression was significantly increased by all transduction routes (topical, intraflap injection, intravascular) ( Po0.01) at day 1, but not at day 7. The spread of luciferase expression was significantly different between the 4 groups at 1 day ( P ¼ 0.026) and was greatest for flaps transduced by intravascular injection. For adenoviral transduction, total radiance was significantly different between the transduced groups at 1, 14 and 28 days (Po0.05 for all comparisons). The highest levels of radiance were seen in the intravascular group. There was a statistically significant difference in the spread of light emission between the 3 groups at 1 ( P ¼ 0.009) and 14 (P ¼ 0.013) days, but this was no longer evident at 28 days. Intravascular adenoviral delivery yields high-level, diffuse and durable gene expression in rat SIE flaps and is suitable for examination in therapeutic models.

Adenovirally Delivered Enzyme Prodrug Therapy with Herpes Simplex Virus–Thymidine Kinase in Composite Tissue Free Flaps Shows Therapeutic Efficacy in Rat Models of Glioma

“…Time course studies demonstrated a significant reduction in adenoviral gene (GFP) expression at 1 and 3 weeks (figures 2B and 2D) (p < 0.0001) and, in relative terms, was found to have decreased by 58% at 1 week and 84% by 3 weeks compared to the 72 hour time point. This finding is expected due to the transient nature of adenoviral gene expression and validates our earlier work, (13) which showed that adenoviral gene expression typically ceases by 28 days following flap infection. Using RT-QPCR we quantified viral TK expression within flap tissues ( figure 2E) and observed significantly elevated levels of TK mRNA tissues of infected SIEA flaps compared to controls (p<0.05).…”

“…Previous work from our laboratory (13), has shown that the optimal route for introducing a viral vector into a free flap is by intra-arterial injection and that introduction of adenoviral vectors in this manner results in diffuse and durable transgene expression within the flap for up to 28 days. Many therapeutic gene therapy strategies have been proposed for use in conjunction with free flaps (12) and, in this paper, we report the pre-clinical evaluation of a virally directed enzyme prodrug therapy (VDEPT) strategy in the microscopic and macroscopic residual disease settings.…”

“…Our flap transfection protocol has been described previously (13) but, in brief, superficial inferior epigastric artery (SIEA) flaps (4 × 2 cm) were marked on the caudal abdomen of Fischer (F344) male rats (250 g) as shown and raised on the SIE vessels under inhalational anaesthesia (Abbott Laboratories, UK). The common femoral (CF) vessels were stripped of adventitia and divided proximal and distal to the origin of the SIE pedicle.…”

“…We have previously shown that the intra-vascular administration of viral vectors into flaps achieves greater transfection efficiencies than nonviral and percutaneous modalities of delivery (13), although topical viral administration has been successful in other contexts (14)(15)(16). Free flap-mediated immunostimulatory approaches (10) to eradicate microscopic residual disease have previously been described, for example, through the over-expression of interleukin-12 (IL-12) (17)(18)(19)(20)(21)(22) and have shown retardation of tumour growth by therapeutic flaps.…”

Locoregional intravascular viral therapy of cancer: precision guidance for Paris's arrow?

“…As perfusion is not limited to organs but rather to an area with a defined inflow and outflow, free tissue flaps, which are commonly used by plastic and other surgeons in the reconstructive process after radical surgery, are also potential targets for intravascular therapy 26 A further theoretical attraction of this approach is that transduction of a flap with virus may provide a mechanism for local action on microscopic residual disease beyond the flap at the tumour bed and, hence, reduce local recurrence. Overall, animal studies have shown that the effective concentrations of a chemother-apeutic agent achievable within target organs by perfusion-type systems are typically 15–25 times those tolerated in systemic administration, 27–31 in which dosages are limited by systemic toxicity.…”